Generally speaking, this invention relates to assemblies for separating blood serum or plasma from the cellular portion of a blood sample. More particularly, this invention relates to a method for sterilizing the assembly by the use of radiation sterilization.
Separation assemblies of the kind discussed herein typically include an elongated glass tube of varying design and containing the blood sample, with the tube being subjected, once the sample is introduced into the tube, to centrifugation until the cellular portion (heavy phase) and serum or plasma (light phase) are completely separated. To maintain separation after centrifuging and to facilitate decanting or removal of the light phase by pipette, a barrier means is provided within the assembly between the phases. It is to this barrier means in particular that the invention herein is directed.
For example, thixotropic gels have been utilized as a barrier medium for such assemblies with the gels responding to centrifugal forces during centrifugation to move into a position separating the heavier and lighter phases of a sample under consideration. Moreover, newtonian gels have been used instead of thixotropic gels. Also, a density gradient medium, namely sucrose/epichlorohydrin polymer with sodium diatrizoate (ficoll-hypaque) has been used alone or in combination with the above-noted gels. Other such products include iodine containing contrast mediums such as NYCODENZTM.TM., owned by Nyegaard & Co., A/S (Norway Joint Stock Company), Oslo 4, Norway. One well-known ficoll-hypaque product is HISTOPAQUER.RTM. a product of Sigma Chemical Co., 3050 Spruce Street, St. Louis, Mo. 63103, while another is FICOLL-PAQUER.RTM. a product of Pharmacia Fine Chemicals AB, Uppsala, Sweden.
At any rate, when it was discovered that ficoll-hypaque could be used with a newtonian or thixotropic gel to have an improved product over either one of these materials separately, the product was presterilized by autoclaving. Alternatively, the ficoll-hypaque may be sterilized by filter sterilization through a, for example, 0.22 micron pore size filter. Both autoclaving and filtering are approaches which accomplish proper sterilization. However, these procedures are somewhat difficult to handle in the sense of manufacturing many thousands of these blood separation tube assemblies in a production line. It is far more convenient, for example, to utilize radiation sterilization which can be imparted in the production line without any separate stage or application as is required for filtering or autoclaving.
It has been found, however, that with the use of ficoll-hypaque in any of the combinations discussed above for blood separation tube assemblies, that a portion of the ficoll-hypaque component precipitates out upon exposure to, for example, Cobalt 60 radiation. Thus, it has been standard procedure to utilize the filtration form of sterilization discussed above, or autoclaving even with the problems noted.
By contrast, it has now been found, with this invention, that radiation sterilization can be used with such tube assemblies, including a ficoll-hypaque product in combination, for example, with a thixotropic gel by the addition of sodium bicarbonate or other buffering material for the density gradient medium component in order to establish a particular pH level after exposure. When this is done, no precipitation takes place. Radiation sterilization is appropriate, therefore, in a production line, in accordance with this invention, for producing tube assemblies of the kind discussed above.
For example, as one specific arrangement of tube assembly, in accordance with this invention, a conventional glass tube may be selected and a thixotropic gel introduced into and adjacent the closed end of the tube. Subsequently, a HISTOPAQUER.RTM. material is introduced on top of the thixotropic gel. In this connection, the HISTOPAQUER.RTM. is, as discussed above, a sucrose/epichlorohydrin polymer with sodium diatrizoate. The sodium bicarbonate buffer is introduced as a 0.1-0.5 percent solution by volume which provides a range of between about 6.5-7.6 pH after radiation exposure.
In considering generally the conditions for carrying out this invention, the buffer may include, for example, sodium bicarbonate or sodium carbonate alone or in combination with potassium or sodium-mono or dibasic phosphates. Another example of buffer which may be utilized, in accordance with this invention, is tris-hydroxymethyl amino methane (TRIS) with or without sodium or potassium phosphate or hydroxyethyl piperazine/N/2/ethane sulfonic acid (HEPES) with or without sodium or potassium phosphate. Phosphate buffers are especially useful for eliminating undesirable pH shifts before and after the sterilization procedure herein because they stabilize the other organic or inorganic buffers present.
It should be borne in mind that when certain of these buffers are selected, in accordance with the invention, the ficoll-hypaque solutions may have to be adjusted at a higher pH of, for example, about 8.5 before radiation sterilization in order to achieve the final desired pH of within the range of between about 6.5-7.6 after sterilization, with a preferred physiological pH being 7.4.+-.0.1. A source of radiation may be Cobalt 60 or by electron beam.
Thixotropic gels of the kind used in the cellular separation of blood as with the assemblies of the invention here are generally described by A. A. Luderer, A. R. Zine, D. M. Hess, J. N. Henyan and G. Odstrchel (Rapid, Quantitative Human Lymphocyte Separation And Purification In A Closed System), Molecular Immunology, 16, pp. 621-624 (1979). Also, U.S. Pat. No. 4,190,535 describes suitable thixotropic gels and their preparation and is incorporated herein by reference in its entirety.
Generally, a water insoluble, thixotropic gel chemically inert to blood constituents which can be used in accordance herewith is formulated from a dimethylpolysiloxane and a precipitated methylated silica in which the methylation renders the material hydrophobic. The thixotropic gel preferably has a specific gravity of within the range of between about 1.055 and 1.080 g/cm.sup.3, and most preferably, formed with a specific gravity of about 1.077g/cm.sup.3. The ficoll-hypaque used, in this invention may have a specific gravity, for example, of 1.09 g/cm.sup.3.
Other objects and advantages of this invention will be apparent from the following description, the accompanying drawings, and the appended claims.